Gloria Jih
Biophysics 205 Semester Proposal
Title: Global analysis of messenger RNA (mRNA) transported by molecular motors.
Abstract: One strategy the cell uses to regulate gene expression is by targeting mRNA to its proper
location prior to translation. It is well documented that molecular motors transport mRNA-bound
protein complexes along the cytoskeleton to subcellular locations such as neuron growth cones and
growing edge of fibroblast cells. Although a few examples have correlated transported mRNA with the
responsible motors, it is not clear whether different types of motors transport different sets of mRNA.
In this proposal, mRNA associated with kinesin-1, kinsin-2, and myosin will identified using
immunoprecipitation and microarray, and verified using in situ hybridization in CAD cells. Structural
sequence of 3' UTR of mRNA associated with specific motors will also be analyzed.
Introduction:
Messenger RNA is synthesized in the nucleus, exported into the cytoplasm, and localized to
either the endoplasmic reticulum (ER) or other subcellular locations. Localization of mRNA to
subcellular locations is used to regulate gene expression and has been observed in various organisms.
In Drosophila embryogenesis, at least 70% of genes encode subcellularly localized mRNA (Lecuyer et
al., 2007). The correct localization of these mRNA is important for the establishment of anteriorposterior axis. In the budding yeast, Ash1 mRNA is delivered by myosin along actin filaments from the
mother cell into the budding daughter cells. This event results in asymmetric distribution of Ash1
protein, thereby controls mating-type switching (Long et al., 1997).
In mammalian cells, mRNA is typically transported along cytoskeleton in granules. These large
protein complexes contain different proteins, including proteins that bind to mRNA, motor proteins that
transport the granules, proteins that inhibit translation, and adaptor proteins. In many cases, the 3' UTR
of mRNA encode specific sequence "zipcode" that forms secondary structure, which serves as docking
sites for RNA-binding proteins (RBPs). These proteins probably serve not only as scaffold, but also as
factors inhibiting translation initiation until the mRNA is transported to the correct location.
In yeast, myosin moving along actin filaments seems to be the only mechanism for mRNA
transport. In higher eukaryotes, kinesin and dynein motors moving along microtubules plays the major
role. For example, kinesin-1 is responsible for the transport of myelin basic protein (MBP) mRNA in
oligodendrocytes (Ainger et al., 1993) and the enrichment of calcium/calmodulin-dependent protein
kinase II (CaMKII) in hippocampal dendrites (Kanai et al., 2004); in xenopus oocytes, kinesin-2
mediates the transport of Vg1 mRNA to vegetal cortex (Betley et al, 2004). Although the role of
microtubule-based motor is evident in mRNA localization, it is not clear what mRNAs are transported
by different motors, and whether different motors carry different sets of mRNA. What are the mRNA
transported by kinesin-1? Does kinesin-2 transport the same set of mRNA as that of kinesin-1?
One way to address these questions is to identify all mRNA bound by a specific RBP, and figure
out which motor transports such RBP. Although this approach would provide insights into the
relationship between all components in the localization complex, it is not practical because the complex
is extremely complicated: One mRNA might be bound by multiple RBPs, and multiple RBPs might be
in the same granule. Another way is to ignore the RBPs and focus directly on the relationship between
mRNA and motors. Thus, in Specific Aim 1, I will describe an experimental method in attempt to
identify all mRNA associated with kinesin-1 and kinesin-2 in neuronal cells undergoing differentiation.
Originally, I had proposed to identify all mRNA transported via microtubule-based motors by
first disrupting the integrity of microtubule, then visualizing mRNA mislocalization via in situ
hybridization. I later determined such large scale experiment is not worthwhile as results will only
show what types of mRNA are localized to subcellular locations via microtubule-based transport. Also,
such experiment would require the synthesis of a genome-wide library of probes, and subsequent
staining of cells using individual probes. Although experimental procedures could be automated, it is
very difficult to get great resolution by in situ hybridization at single cell level. To reduce the amount of
time and labor, only candidates identified in Specific Aim 1 will be further validated in Specific Aim 2
using in situ hybridization. In Specific Aim 3, I will analyze 3' UTR sequence of mRNA transported by
the same motor protein to identify sequences that might confer indirect specificity to motor protein.
Specific Aim 1: To identify mRNA transported by kinesin-1 or kinesin-2 using immunoprecipitation
and DNA microarray.
CAD cells express neuron-specific proteins and undergo morphological differentiation upon
removal of serum from tissue culture media. Processes from differentiated cells contain abundant
parallel microtubules, actin filaments, large granules and small vesicles. I expect kinesin-1 and kinesin2 to play a significant role in transporting mRNA to growing processes upon serum removal.
CAD cells grown in media containing serum will be transfected with plasmids containing HAkinesin-1 or HA-kinesin-2. After transfection, cells will be induced to differentiate in media without
serum, lysed and incubated with anti-HA antibody and subsequently with Protein-A sepharose. RNA
will be extracted and biotinylated cRNA probes will be synthesized. Probes will be used for
hybridization to Affymetrix GeneChip. For control, I will use non-transfected cells.
For comparison, I will isolate and hybridize RNA from transfected cells that will not have been
allowed to undergo differentiation. These cells should not have processes, thus I expect kinesin motors
to be associated with less mRNA or a different set of mRNA.
In order to assess the specificity between mRNA and motors, I will also transfect cells with
plasmids containing HA-myosin. Then cells will be allowed to undergo differentiation. mRNA
associated with myosin will be isolated and analyzed. Since myosin is known to move and transport
molecular cargoes along actin filaments in yeast, it would be interesting to see whether myosin
transport mRNA in mammalian cells and whether the transported set of mRNA is similar or different
from that of kinesin motors.
Specific Aim 2: To verify mRNA candidates associated with microtubule-based transport.
As mentioned in the introduction, I will evaluate whether mRNA candidates from Specific Aim
1 are specifically localized to processes upon differentiation induction. I will first culture nontransfected CAD cells in differentiating media, then Taxol will be added to interfere with the growth of
microtubule in cellular processes. I will hybridize fluorescent probes designed to anneal to candidate
mRNAs, and visualize mRNA localization pattern using microscopy. For control, I will hybridize
probes to differentiated cells without Taxol treatment. Because perturbation of microtubule will result
in disruption of microtubule-based transport, I expect the localization pattern of candidate mRNA to
change when cells are treated with Taxol.
I will also hybridize probes generated for myosin-associated mRNA in Taxol-treated cells. Since
Taxol only perturbs microtubule-based transport, localization of these mRNA to cellular processes
should not change.
Specific Aim 3: To analyze 3' UTR of candidate mRNA for sequences that might confer indirect
specificity to motor protein.
I will examine sequence and structural homology of each set of motor-specific mRNA. It is very
likely that sequence homology is not as crucial as structural homology, so efforts will be made to yield
correct prediction of the secondary structure of mRNA.
This specific aim is especially challenging considering many factors are involved in the
transport complex. Since motor protein do not bind directly to mRNA, this attempt will at most yield
indirect association between mRNA and its respective motor. Therefore, structural analysis is
insufficient to determine by which motor protein the mRNA will be transported. However, it might be
useful and necessary to potentially characterize sets of motor-specific mRNA. Known relationships
between mRNA sequence, corresponding RBP, and motor can be used as prototype in the computation
process. For example, 3' UTR of CamKII_alpha mRNA contains a cytoplasmic polyadenylation
element (CPE) that is bound by CPE-binding protein (CPEB), which is found in granules containing
both kinesin and dynein (Huang et al., 2003).
Conclusion: Bidirectional granules are found to contain both kinesin and dynein motors. However, it
has not been shown that one granule could contain more than one type of kinesin motor or contain
kinesin and myosin simultaneously. It is very possible that different motors transport different subsets
of mRNA, resulting in asymmetric distribution of protein within a cell.
Reference:
Ainger et al. (1993) Transport and localization of exogenous myelin basic protein mRNA microinjected
into oligodendrocytes. J. Cell Biol. 123, 431.
Betley et al. (2004) Kinesin II mediates Vg1 mRNA transport in Xenopus oocytes. Curr. Biol. 14, 219.
Huang et al. (2003) Facilitation of dendritic mRNA transport by CPEB. Genes Dev. 17, 638.
Kanai et al. (2004) Kinesin transports RNA isolation and characterization of an RNA-transporting
granule. Neuron 43, 513.
Lécuyer et al. (2007) Global analysis of mRNA localization reveals a prominent role in organizing
cellular architecture and function. Cell 131, 174.
Long et al. (1997) Mating type switching in yeast controlled by asymmetric localization of ASH1
mRNA. Science 277, 383.